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??OBJECTIVE To prepare the tulobuterol crystal reservoir patch, and to evaluate morphology, stability and crystallization factors of the crystal in the patch, adhesive force, dissolution, transdermal properties in vitro and the pharmacokinetics in rabbits. METHODS The transdermal patch was prepared on the basis of drug recrystallization and characterized by morphology, stability and crystallization factors using microscope and adhesive force using initial adhesion tester, adhesion tester and peel tester. The dissolution and transdermal properties were evaluated by using the dissolution tester and transdermal tester. In addition, pharmacokinetics was studied using New Zealand rabbits as experimental animals. RESULTS The drug crystals were evenly distributed in the form of filaments, which had average width of (4.4??1.8)??m and kept stable at 2-40 ??. The crystallization in patches is affected by tulobuterol supersaturation and temperature. The adhesive force of patch was suitable and its dissolution matched standard which can be fitted by the Higuchi equation. In the diffusion cell in vitro, the drug penetrated through the skin in a Zero-order kinetic equation, and the cumulative penetration percentage and skin retention concentration were 92.04% and 10.36 ??g??cm^-2 with in 24 h. The pharmaceutic parameters showed that the tulobuterol blood concentration can be maintained within 24 h, whose t_max and ??_max were (6.67??3.06)h and (3.08??1.32) ng??mL^-1, respectively. CONCLUSION The tulobuterol crystal reservoir patch can be established by control of recrystallization conditions. The patch has good adhesive properties and sustained release characteristics in vitro and in vivo, which has the practical significance for further study.     

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??OBJECTIVE To enhance dissolution rate of silymarin (SM) by forming SM nanoparticles (SM-NA) with supercritical anti-solvent (SAS) method. MOTHODS Single-factor test was employed to investigate the influencing factors of particle size and yield of SM-NA, such as pressure, temperature, flow rate, and concentration of the solution. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and differential scanning calorimetry (DSC) were used to determine the state of SM-NA. The dissolution characteristics in vitro were investigated with 0.3% SDS aqueous solution as the solvent and compared with the SM powder. RESULTS Taking size distribution as well as yield of SM-NA as the evaluation indexes, the optimal prepration parameters were selected as followings:pressure 15 MPa, temperature 35 ??, flow rate 1.5 mL??min^-1, concentration 100 mg??mL^-1. The XRD and DSC of SM-NA described the decrease of SM in crystallinity, and it was transformed mostly with the amorphous state, compared with the SM powder. The accumalte release rate of SM-NA achieved 80% within 10 min, markedly higher than that of the SM powder and its commercial preparation. CONCLUSION The SM-NA prepared by SAS has remarkablly smaller particle size thus can greatly improve the in vitro release of SM.
     

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??OBJECTIVE To prepare lappaconitine(LA)-loaded chitosan/ sodium ??-glycerophosphate(CS/??-GP) thermosensitive hydrogels and investigate its phase transition mechanism of gel formation process and release properties in vitro. METHODS The injectable CS/??-GP thermosensitive hydrogels were prepared with biodegradable CS as carrier material and ??-GP as coagulation accelerator. The release behavior in vitro was studied by dynamic dialysis, and the phase transition mechanism of gel formation process was further investigated by rheological method. RESULTS The optimized process condition was as follows:the concentration of ??-GP and CS was 560 and 22 mg??mL^-1, respectively, CS was dissolved by 0.1 mol??L^-1 HOAc, and the valume ratio of CS to ??-GP was 8.75??1.25(V/V), the gelation time of CS/??-GP thermosensitive hydrogels with volume ratio of 8.75??1.25(V/V) at 37 ?? was 5 min 38 s. The in vitro release study showed that these injectable CS/??-GP thermosensitive hydrogels had sustained release effect for LA, and the release behavior could be well described by the Higuchi model and Korsmeyer-Peppas model. The mechanism of LA releasing from CS/??-GP thermosensitive hydrogels was attributed to drug dissolution and diffusion. Rheological studies showed that the CS/??-GP thermosensitive hydrogels belonged to thixotropic system and exhibited non-Newtonian and shear-thinning fluid behavior as well as ??solid-like?? gelatin behavior. CONCLUSION LA-Loaded CS/??-GP injectable thermosensitive hydrogels with good elasticity and gel strength properties are prepared successfully, and they show sustained release effect of LA in vitro.     

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??OBJECTIVE To clone target gene by RT-PCR method, construct VEGF₁₆₅ lentivirus vectors, transfect adipose tissue derived stem cells (ADSCs) and verify the expression of VEGF₁₆₅ in vitro and in vivo. METHODS RT-PCR technology was employed to clone VEGF₁₆₅ gene, and this gene was cloned to lentivirus vector pLVX-EF1??-IRES2-AcGFP1 to construct a lentiviral vector pLVX-EF1??-VEGF₁₆₅-IRES2-AcGFP1. Bacterial colonies PCR and sequencing analysis were used for identification. Then, Lenti-X 293T cells were transfected with main vector pLVX-EF1??-VEGF₁₆₅-IRES2-AcGFP1, packaging plasmid gag-pro, vpr-pol, Tet-Off^TM, tat-IRES-rev and coating plasmid env(VSV-G). Lentiviral vectors were packaged and the titer was determined. ADSCs were isolated by collagenase digestion method, then cultured, and identificated by morphology, immunofluorescence and multi-directional differentiation. ADSCs was transfected with packaged VEGF₁₆₅ lentivirus. Immunofluorescence and ELISA were used to detect the expression of VEGF₁₆₅ in vitro. ADSCs transfected with VEGF₁₆₅ lentivirus were injected into nude mice. ELISA was used to detect the expression of VEGF₁₆₅. RESULTS The VEGF₁₆₅ gene fragment was cloned successfully, and the lentiviral vector plasmid pLVX-EF1??-HVEGF₁₆₅-IRES2-AcGFP1 was confirmed to contain VEGF₁₆₅ gene fragment as shown by bacterial colonies PCR. DNA sequencing analysis confirmed that VEGF₁₆₅ gene sequencing was exactly the same with that reported by Genbank. After transfection, a large number of Lenti-X 293T cells with green fluorescence were observed by fluorescent microscopy. The concentration of the virus titer was 1??10⁸ TU??mL^-1. ADSCs were identified by morphology, immunofluorescence and multi-directional differentiation methods, in line with the literature reported ADSCs characteristics. There were about 90% of ADSCs which could express VEGF₁₆₅ after being transfected with the viruses by immunofluorescence detection, also, VEGF₁₆₅ protein was proved by ELISA to express stably and efficiently in vitro and in vivo. CONCLUSION Lentiviral vectors expressing VEGF₁₆₅ are successfully constructed by cloning target gene with RT-PCR method. After transfection, ADSCs expressing VEGF165 protein stably in vitro and in vivo can be obtained.
     

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??OBJECTIVE To establish a measuring method for microdialysis probe recovery of ginsenoside Rg₁ and investigate the effects of flow rate, concentration and using times of probe on the recovery in vivo and in vitro. METHODS Dialysis method and retrodialysis method were used for the study. The concentration of ginsenoside Rg₁ in brain and blood dialysate was determined by LC-MS/MS and the probe recovery was calculated. RESULTS The recoveries of brain and blood microdialysis probes showed good stability within 10 h, with average values of 17.0% and 34.4% respectively for ginsenoside Rg₁ at 1.5 ??L??min^-1. Concentrations (50,200,500,1 000 ng??mL^-1) had no obvious effect on recovery. At the same concentration, the recovery of brain and blood probes for ginsenoside Rg₁ decreased with the increase of flow rate (0.5, 1.0, 1.5, 2.0, 3.0 ??L??min^-1) in vitro and in vivo. The dialysis recoveries of brain and blood probes in vitro were (40.6??4.3)%, (23.5??2.3)%, (17.7??0.8)%, (12.2??1.1)%, (8.8??0.6)% and (70.6??3.6)%, (46.0??2.1)%, (32.9??1.6)%, (25.6??0.7)%, (18.2??1.3)%, respectively. The recoveries of dialysis and retrodialysis in vitro were approximately equal, and the recovery detected by retrodialysis in vivo was similar with the in vitro results. Probe used for no more than 3 times still kept high transmittance by flushing with 2% heparin sodium and ultrapure water successively. CONCLUSION Retrodialysis method can be used to study brain and blood probe recovery in vivo, and microdialysis can be used for simutaneous pharmacokinetic studies of ginsenoside Rg₁ in intercelluar fluid and blood.     

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??OBJECTIVE To prepare ion-sensitive ophthalmic in situ gel containing bendazac lysine (BDZL-ISG) and preliminarily study its rheological behavior, in vitro drug release, corneal permeation, and pharmacokinetics in rabbit aqueous humor. METHODS Single factor investigation was carried out to optimize the formulation, taking viscosity and gelling capacity as evaluation indices. Using aqueous solution or eye drops as control, the in vitro release of the formulation was evaluated by dialysis membrane method. Then, the corneal permeation experiment of the optimum formulation was carried out with Franz diffusion cell. The pharmacokinetics of BDZL-ISG in rabbit aqueous humor was preliminarily studied by microdialysis. RESULTS Compared with the control group, the optimum formulation had shear thinning behavior and significant sustained release effect. There was no significant difference in the corneal permeation between the two groups. The RESULTS of pharmacokinetic study showed that ??_max (13.25 ??g??L^-1) and AUC_0-t of BZDL-ISG were 2.38 and 2.2 times higher than those of BDZL eye drops respectively, which suggested that the ocular bioavailability of BDZL was greatly enhanced by the optimum in situ gel formulation. CONCLUSION With significant sustained release effect, the ion-sensitive ophthalmic in situ gel will become a promising alterative formulation for bendazac lysine for treatment of cataracts.     

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??OBJECTIVE To evaluate the effects of drug concentration and perfusion rate on the recoveries of self-made linear microdialysis probes for further ocular pharmacokinetic study. METHODS Brimonidine tartrate was selected as the model drug. The in vitro recovery was determined using positive dialysis and retrodialysis at different perfusion rates and drug concentrations. And the in vivo recovery was determined using retrodialysis method. RESULTS The microdialysis recoveries of brimonidine tartrate were inversely proportional to perfusion rate,while independent of drug concentration. The positive dialysis and retrodialysis recoveries in vitro were different at 1.0 ??L??min^-1, but no significant difference at 2.0 and 3.0 ??L??min^-1. The in vitro recoveries were greater than those in vivo. CONCLUSION The self-made microdialysis probe has stable recovery and can be used in ocular pharmacokinetic study of brimonidine tartrate.     

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??OBJECTIVE To prepare docetaxel liposomes by freeze-drying of tert-butyl alcohol (TBA)/water cosolvent system and evaluate the in vitro and in vivo properties. METHODS The formulation was optimized by single factor screening experiments. The effects of TBA/water ratio, lipid/drug ratio, sucrose/lipid ratio and mixing temperature on encapsulation efficiency were investigated. The in vitro release profiles were also investigated with docetaxel injection(Duopafei^?k) as control. RESULTS The formulated liposomes had a mean size of 263 nm with entrapment efficiency of (88.45??1.63)% at TBA/water ratio 50??50, lipid/ drug ratio 10??1, sucrose/lipid ratio 5??1 and mixing temperature 60 ??. In vitro drug release from liposomes lasted for 48 h. CONCLUSION Freeze-drying of TBA/water cosolvent system method may be feasible to prepare docetaxel liposomes.